Abstract: Basal cell derived salivary and breast cancers are correlated with poor patient prognosis, increased cancer reoccurrence and lack of definitive therapies1. In both branching organs, cytokeratin 5-positive (K5+) basal cells contribute to ductal cells during normal development2?4 and recent research has highlighted similarities in the K5+ basal cell origin of mammary and salivary squamous cells5,6. In mammary tumors of basal origin, irradiation- resistant cancer stem cells (CSCs) demonstrate K5 positivity1. Both Vitamin D and Vitamin A signaling pathways have been implicated in prevention of tumorigenesis7,8. In the developing9 salivary gland, we and others10 have recently demonstrated that retinoic acid receptor (RAR) signaling contributes to expansion of K5+ basal cells, a phenomena conserved in the adult salivary gland11. In both mammary12 and salivary glands13, CSCs display an upregulated level of ALDH, an aldehyde dehydrogenase that oxidizes Vitamin A to retinol, the precursor to retinoic acid and signaling through RARs. The relationship between RAR signaling and K5+ basal cells during tumorigenesis has yet to be explored. Work in the Welsh laboratory has shown that deletion of the Vitamin D receptor (VDR) in mice leads to increased cell proliferation and tumorigenesis in the breast7 where VDR activity is linked to K5+ cell fate transition4. In human population studies, low levels of Vitamin D are associated with increased cancer incidence and progression14?16. In the submandibular salivary gland (SMG), progenitor cell populations are highly positive for VDR17 and in humans, VDR localizes to epithelial cells18; yet, Vitamin D signaling has not been specifically explored in the salivary gland. We will test the hypothesis that co- activating VDR and RAR signaling will reduce K5+ basal cell expansion and tumorigenesis in the SMG. In this proposal we will address the following questions: 1) What is the role of VDR signaling in K5+ basal cell expansion and differentiation in the SMG? 2) Does VDR signaling synergize with RAR? signaling to negatively regulate K5+ basal cell expansion in the SMG? 3) Will loss of VDR signaling synergize with inhibition of RAR? signaling to enhance K5+ tumorigenesis in the SMG? 4) Will activation of VDR and RAR? signaling in K5- TdTomato-positive tumor cells using directed delivery of vitamin derivatives and small molecules decrease tumorigenic cell properties? To determine if RAR? and VDR signaling synergize in negatively regulating the K5+ population in the SMG, in Aim 1 we will inhibit RAR? signaling in a VDR knockout K5 reporter mouse and trace K5+ basal cell fate. In Aim 2, we will determine if VDR and RAR? pathways synergize to regulate K5+ cell expansion in basal salivary tumors. This aim will be achieved with targeted pharmacological inhibition of RAR? signaling in a VDR knockout K5 reporter mouse induced for tumorigenesis with DMBA. Additionally, we will determine if activation of VDR and RAR? signaling in K5-TdTomato-positive tumor cells using small molecules and vitamin derivatives in 3D culture will affect cell division and motility.